Bronchiolitis is a severe lower-respiratory tract infectious disease primarily caused by members of the Paramyxoviridae family. Human respiratory syncytial virus (hRSV) is the principal cause of morbidity in children less than 2 years of age as well as the elderly, immuno-compromised and transplant patients. To date, there are neither vaccines nor efficacious approved drugs to prevent or treat hRSV infection. The immuno-prophylactic antibody palivizumab is approved for high-risk patients only such as premature babies and infants suffering from underlying diseases. The broad-spectrum small molecule antiviral ribavirin is available to treat infection, but it has considerable side-effects and limited efficacy. During the past decade, a number of drug candidates targeting hRSV entry or replication steps have been advanced to pre-clinical or clinical development.
The hRSV genomic RNA (vRNA) is packaged by the viral nucleoprotein (N) at all times, forming a N:RNA complex, called nucleocapsid. This ribonucleoprotein complex is used as a template for mRNA transcription and genomic or antigenomic RNA replication by the RNA-dependent RNA polymerase (RdRp), which is composed of 2 major viral proteins: the phosphoprotein P and the large polymerase L. In this complex, the phosphoprotein is an essential co-factor of the L polymerase by binding to L and N and targeting the polymerase L to vRNA. Two co-factors, M2-1 and M2-2, are required for the RdRp to process RNA efficiently during the viral cycle. M2-1 is a tetrameric transcription processivity factor that binds in a competitive manner to RNA and P via its core domain. M2-1 functions as an anti-terminator of transcription that prevents premature termination of transcription both intra- and inter-genetically. Although in vitro experiments have shown that M2-1 binds preferentially to positive-sense viral gene end (GE) and poly-A sequences, the exact mechanisms by which M2-1 improves transcription efficiency is not fully understood.
Recently, cyclopamine (CPM) and jervine were identified as highly potent and selective inhibitors of hRSV replication in vitro. CPM is a well-known antagonist of the smoothened protein (Smo) receptor, a 7-transmembrane receptor of the Sonic hedgehog signaling pathway (Shhp). The Shhp is involved in embryonic development, cell differentiation and tumorigenesis. Cyclopamine is a naturally occurring chemical that belongs to the group of steroidal jerveratrum alkaloids. It is a teratogen isolated from the corn lily (Veratrum californicum) that causes usually fatal birth defects. It can prevent the fetal brain from dividing into two lobes (holoprosencephaly) and cause the development of a single eye (cyclopia). It was caused by inhibiting the hedgehog signaling pathway (Hh).
The teratogenic properties of cyclopamine largely preclude its development as a pediatric drug for the treatment of RSV infections. These properties could lead to serious adverse events in treated patients.
Invention
In order to overcome the teratogenic property of CPM, and to pad the internal space of clinical RSV infection treatment, specially pediatric treatment, the present invention provides a kind of compound for RSV infection treatment and its preparation method and application. The compound described can inhibit RSV replication, and cannot inhibit Hedgehog signaling pathway. Therefore, the compound will be a sage and effective anti-RSV drug for clinic, relieving of teratogenicity.
To access to the goals above, the following technical scheme was designed for the present invention:
A kind of compound to treat RSV infection, which is CPM's chemical analogue, with inhibition property on RSV replication, without inhibition property on Hedgehog pathway.
The compound described is a kind of novel chemical entity, including S1-2, S1-4, and all of the possible cyclopamine chemical analogues, with inhibition of respiratory syncytial virus replication effect and without inhibition of the Hedgehog signaling pathway effect. S1-2 and S1-4 is the two instances therein.
The serial of chemical analogues of CPM structure was published in the Chinese patent CN101631463A. And the same analogues were recorded in American patent US20080293754A1.
A typical CPM structure was showed in formula 1.

A kind of optimized CPM analogue was showed in formula I,

In the formula, R1 is H, OH, alkyl group, sulfonamide group, sulfonyl amino, —OC(O)R5. —N(R5)C(O)R5 or glycosyl group;
R2 is H, alkyl group, alkenyl group, alkynyl group, naphthenic base, nitrile group, or heterocyclic alkyl; Or, R1 binds with R2, to form ═O, ═S, ═N(R), ═N(NR2) or ═C(R)2;
R3 is H, alkyl group, alkenyl group or alkynyl group;
R4 is H, alkyl group, alkenyl group, alkynyl group, naphthenic base, heterocyclic alkyl, aralkyl, ceteroary, alkyl aromatic, halogenated alkyl, —OR5, —C(O)R5, —CO2R5, —SO2R5—C(O)N(R5)(R5), —[C(R)2q—R5, —[(W)—N(R)C(O)]qR5, —[(W)—C(O)]qR5, —[(W)—C(O)O]qR5, —[(W)—OC(O)]qR5, —[(W)—SO2]qR5, —[(W)—N(R5)SO2]qR5, —[(W)—C(O)N(R5)]qR5—[(W)—O]qR5, —[(W)—N(R)]qR5, —W—NR53+X— or —[(W)—S]qR5;
In which, W is divalent alkyl independently;
R is H or alkyl separately;
q is 1, 2, 3, 4, 5, or 6 independently;
X— is a halogen;
R5 is H, alkyl group, alkenyl group, alkynyl group, aryl group, naphthenic base, heterocyclic alkyl, aralkyl, ceteroary, alkyl aromatic, or —[C(R)2]p—R6; P is 0˜6; Or any two R5 on the same substituent group can form a 4˜8 member ring containing 0˜3 miscellaneous atoms, and the heteroatoms are selected from N, O, S or P; The R6 is independently hydroxyl, —N(R)COR, —N(R)C(O)OR, —N(R)SO2(R), —C(O)N(R)2, —OC(O)N(R)(R), —SO2N(R)(R), —N(R)(R), —COOR, —C(O)N(OH)(R), —OS(O)2OR, —S(O)2OR, —OP(O)(OR)(OR), —NP(O)(OR)(OR) or —P(O)(OR)(OR). The limiting condition is that R1 is not hydroxyl or glycosyl group when R2, R3 and R4 is H. Another limiting condition is that R1 and R2 are not C═O when R4 is hydroxyl.
For one selected optimized CPM analogue compound, as shown in formula II,

R1 is H, OH, alkyl group, amidogen, sulfonamide, sulfonamido-, —OC(O)R5, —N(R5)C(O)R5 or glycosyl group;
R2 is H, alkyl group, alkenyl group, alkynyl group, aryl group, naphthenic base, nitrile group, heterocyclic alkyl;
R3 is H, alkyl group, alkenyl group, alkynyl group;
R4 is H, alkyl group, alkenyl group, alkynyl group, naphthenic base, heterocyclic alkyl, aralkyl, ceteroary, alkyl aromatic, halogenated alkyl, —OR5, —C(O)R5, —CO2R5, —SO2R5—C(O)N(R5)(R5), —[C(R)2q—R5, —[(W)—N(R)C(O)]qR5, —[(W)—C(O)]qR5, —[(W)—C(O)O]qR5, —[(W)—OC(O)]qR5, —[(W)—SO2]qR5, —[(W)—N(R5)SO2]qR5, —[(W)—C(O)N(R5)]qR5—[(W)—O]qR5, —[(W)—N(R)]qR5, —W—NR53+X— or —[(W)—S]qR5;
In which, W is divalent alkyl independently;
R is H or alkyl separately;
q is 1, 2, 3, 4, 5, or 6 independently;
X— is a halogen;
R5 is H, alkyl group, alkenyl group, alkynyl group, naphthenic base, heterocyclic alkyl, aralkyl, ceteroary, alkyl aromatic, or —[C(R)2]p-R6; P is 0˜6; Or any two R5 on the same substituent group can form a 4˜8 member ring containing 0˜3 miscellaneous atoms, and the heteroatoms are selected from N, O, S or P; The R6 is independently hydroxyl, —N(R)COR, —N(R)C(O)OR, —N(R)SO2(R), —C(O)N(R)2, —OC(O)N(R)(R), —SO2N(R)(R), —N(R)(R), —COOR, —C(O)N(OH)(R), —OS(O)2OR, —S(O)2OR, —OP(O)(OR)(OR), —NP(O)(OR)(OR) or —P(O)(OR)(OR);
In another optimized CPM analogue compound, as shown in formula II, R1 is OH, H, C1˜C3 alkyl group replaced or not;
R2 is C1˜C3 alkyl group, alkenyl group, alkynyl group replaced or not;
R3 is C1˜C3 alkyl group, alkenyl group, alkynyl group replaced or not;
R4 is C1˜C3 alkyl group, alkenyl group, alkynyl group replaced or not;
The “replaced” described means that, one or more H in alkenyl group were replaced with substituent as following, C1˜C10 alkyl group, C1˜C10 alkoxy, hydroxyl, carboxyl, C1˜C10 carbonyl, C1˜C10 acylamino, C2˜C10 ester group, C6˜C30 aryl group, halogen atom, cyanogroup, thioether group;
Another selected optimized compound, R1 is OH, R2 is methyl, R3 is methyl, R4 is methyl.
The compound S1-2 described in formula III, was modified based on formula II, in which R1 and R4 was replaced.
The compound S1-4 was described in formula IV, which was modified based on formula II, R1 and R4 in formula II was replaced.
And the difference between S1-2 and S1-4 is that R1 in formula II was replaced with different substituent group.

A kind of compound preparation method, which was used to treat respiratory syncytial virus infection. The characters of the method are: the chemical synthesis method described to get cyclopamine analogs, and screening analogue by two parallel test in vitro. The described experiments in vitro conducted by two parallel include (1) based on cell experiment, measurement of respiratory syncytial virus replication ability in excitable cells, and (2) based on cell experiment, measurement of a molecular inhibition of sonic hedgehog pathway level, and the inhibition effect is interference level on SAG-induced and Smo-dependent gene transcription.
A method of preparation of compounds for respiratory syncytial virus infection treatment, is characterized by the following steps:
1) Synthetic compounds
2) Screening compounds
The preparation method compounds described, consist of two sub-steps:
1) Preparation of intermediates;
2) Preparation compounds
The procedure of the compound screening includes four sub-steps:
1) The inhibitory effect of test compounds on the respiratory syncytial virus replication
2) The inhibitory effect of the test compounds on the Hh signal pathway
3) Evaluation respiratory syncytial virus replication and Hh activity inhibition coefficient
4) Screening compounds
The preparation procedure of intermediate is as following. First of all, cyclopamine and triethylamine was dissolved in dry dichloromethane DCM; Secondly, under the condition of 0° C., join trifluoroacetic acid anhydride dissolved in DCM into the solution; Thirdly, the reaction mixture slowly heated to a temperature of 15° C. and stirring 16 h. Fourthly, the initial raw materials were confirmed by liquid chromatography-mass spectrometry. Fifthly, concentrate the reaction mixture and dilute the residue with methanol; Sixthly, get the received suspension to be heated to 70° C. and 1 h heat preservation; Seventhly, the reaction mixture cooling to 15° C.; Finally, the intermediate object is white solid by filtration.
The procedure of compounds preparation is as following. First of all, intermediate and triethylamine was dissolved in dry dichloromethane DCM, cooled in ice; Secondly, join trifluoroacetic acid anhydride dissolved in DCM into the solution; Thirdly, the reaction mixture slowly heated to a temperature of 45° C. and stirring 48 h. Fourthly, the initial raw materials were confirmed by TLC. Fifthly, concentrate the reaction mixture in vacuum; Sixthly, clean the residue with methanol; Finally, get the compound object with white solid by filtration.
The detection procedure of compound effect on RSV replication inhibiting is as following. Human respiratory syncytial virus Long strain and HEp-2 cells were used for infection assays. Cells were maintained in DMEM, supplemented with penicillin/streptomycin and 10% FBS, and incubated at 37° C., 5% CO2. Virus was passaged in HEp-2 cells in the same conditions, but with 2% FBS. Virus stocks were prepared by infecting confluent HEp-2 cells at a low multiplicity of infection (MOI) of 0.1 for two to three days. Cells and virus-containing supernatant were then subjected to a single freeze/thaw cycle at −80° C. to release cell-bound virus, and the sample was clarified by centrifugation at 2000×g for 10 min at 4° C. The supernatant was homogenized, aliquoted and stored at −20° C.
The anti-hRSV potency of compounds was evaluated by focus reduction assay following the focus forming assay method, which means that titer calculation was based on the wells with 50 to 100 focus forming. Compounds were tested either during virus adsorption for 1 h at 4° C. to investigate viral entry, post-adsorption for 72 h at 37° C. to investigate viral propagation and replication, or during all stages of infection. The concentration of compound resulting in 50% inhibition (IC50) of virus replication was determined by focus counting (viral entry) or focus size measurement (viral propagation) and was determined using non-linear regression analysis using GraphPad Prism. To assess the cytotoxicity of compounds, a CellTiter-Glo Luminescent Cell Viability Assay was carried following the manufacturer's instruction in the conditions of the focus reduction assay.
The detection procedure of compound effect on Hh signal pathway inhibiting is as following. NIH3T3 cells are seeded cells in 6 well plates, or 12 well plate one day in advance, with 5% FBS DMEM, without antibiotics per well so that they will be 90˜95% confluent at the time of transfection. Plasmids are diluted with Lipofectamine 2000 (total volume is 100-1000 ìl) and added to each well containing cells and medium, followed by incubation at 37° C. in a CO2 incubator for 48 h. After 48 h transfection, SAG is added to reach a final concentration of 0.5 ìM and CPM analogs at various concentrations. At the end of the incubation, cells are washed, lysed and lysate transferred to a 96-well white plate. LAR II reagent is added (from Promega, Dual-luciferase reporter assay system) and samples read in a luminescence reader. Selected compounds from the antiviral RSV assay were tested in the Hh assay in which the ability of the analog to trigger a CPM-like response (inhibition of Smo-dependent SAG induced gene expression).
In the Hh signal pathway inhibition detection assay, the CPM analogs tested is selected from anti-RSV assay and show effective inhibition on RSV replication. With Hh signal pathway inhibition assay, the effect of the CPM analogs on SAG-induced and Smo-dependent gene expression was analyzed, which is similar to CPM, but with various level.
To assess the differential Hh pathway inhibition vs. replication inhibition of analogs (cpd), we determined a coefficient of inhibition (ICrh).
  ICrh  =                                ⁢              Cdp        ⁢                                  ⁢                  (                      %            ⁢                                                  ⁢            RSV            ⁢                                                  ⁢                          inhibition              /              %                        ⁢                                                  ⁢            Hh            ⁢                                                  ⁢            inhibition                    )                    ⁢                                                      ⁢              CPM        ⁢                                  ⁢                  (                      %            ⁢                                                  ⁢            RSV            ⁢                                                  ⁢                          inhibition              /              %                        ⁢                                                  ⁢            Hh            ⁢                                                  ⁢            inhibition                    )                    ⁢                          
In the formula we described, the denominator is the ratio of CPM inhibition effect, which means the ratio of inhibition on RSV to inhibition to Hh. The numerator is the ratio of prepared CPM analog inhibition effect. According to the definition of a coefficient of inhibition (ICrh), if the compound tested is CPM, the coefficient is 1. If the compound tested is an analog with a score lower than 1, indicating that is an unfavorable inhibition profile, eg anti-RSV activity is lower, while Hh inhibition is still strong. A score higher than 1 indicates a favorable profile.
The substep of screening compounds is that selecting the compounds with ICrh data larger than 1, and filtering out the compounds with ICrh data smaller than 1. Furthermore, the compounds with larger ICrh data will be selected preferentially.
The application of compounds is for treatment of respiratory virus infection disease. Briefly, the diseases consist respiratory virus infection, paramyxovirus, respiratory syncytial virus infection, capillary bronchitis/pneumonia/tympanitis caused by respiratory syncytial virus. Furthermore, the compounds never cause side effects on patients, such as fetal anomaly.
The advantage and beneficial effect of the invention is as following:
1) The compounds provided in this invention can be used in the diseases following treatment effectively, including respiratory virus infection, paramyxovirus, respiratory syncytial virus (RSV) infection, capillary bronchitis/pneumonia/tympanitis caused by respiratory syncytial virus.
2) the compounds provided in this invention never cause side effects on patients, such as fetal anomaly.
3) the preparation method provided in this invention can be used to synthesize various kind of compounds for RSV infection treatment, without side effects absolutely.
4) The preparation methods provided in the invention is easy to do, and with wide material source, low production cost, high economic value.
5) The compounds provided in the invention padded the inter space of RSV infection treatment drug, especially for the pediatric drug. Therefor, it is full of important social value.
As shown in the figure, compounds were tested at 2 concentrations (2 ìM, 8 ìM) in the RSV replication assay. The control (compound-free) is scored at 100% replication. Replication in the presence of CPM is scored at 0% replication. Replication inhibition by analogs S2-6 and C2-2 to C2-4 are scored % inhibition of control.
FIG. 5 shows the effect of compounds described in the invention on Smo associated induced gene expression.
Figure shows that compounds were tested at 10 uM in the hedgehog (Hh) assay. The control (compound-free, presence of SAG) is scored at 100% gene expression. The gene expression in the presence of CPM is significantly reduced by 56%. Gene expression inhibition by analogs S1-2 and S1-4 however are not significant, with 36% and 5% respectively. These data show the proof of principle that the anti-RSV activity of steroidal alkaloid molecules derived from CPM can be dissociated from the inhibitory activity of the Smo-dependent Hh pathway.
FIG. 6 shows the Inhibition of coefficient (ICrh) of CPM and its analogs.